Many complex biochemical pathways have now been manipulated genetically, usually by suppression or over-expression of single genes. Further exploitation of the potential for plant genetic manipulation will require the coordinated manipulation of multiple genes in a pathway. A number of approaches have been used to combine transgenes in one plant—including sexual crossing, retransformation, co-transformation, and the use of linked transgenes. A chimeric transgene with linked partial gene sequences can be used to coordinately suppress numerous plant endogenous genes. Constructs modeled on viral polyproteins can be used to simultaneously introduce multiple coding genes into plant cells (for a review, see Halpin et al., Plant Mol. Biol. 47:295-310 (2001)).
Enhancement of gene expression in plants may occur through the introduction of extra copies of coding sequences of the genes into a plant cell or, preferably, the incorporation of extra copies of coding sequences of the gene into the plant genome. Over-expression may also occur through increasing the activities of the regulatory mechanisms that regulate the expression of genes, i.e., up-regulation of the gene expression.
Suppression of gene expression, also known as silencing of genes, in plants occurs at both the transcriptional level and post-transcriptional level. There are various methods for the suppression of expression of endogenous sequences in a host cell. Such methods include, but are not limited to, antisense suppression (Smith et al., Nature 334:724-726 (1988)), co-suppression (Napoli et al., Plant Cell 2:279-289 (1989)), ribozymes (Kohler et al., J. Mol. Biol. 285:1935-1950 (1999)), combinations of sense and antisense (Waterhouse et al., Proc. Natl. Acad. Sci. USA 95:13959-13964 (1998)), promoter silencing (Park et al., Plant J. 9(2):183-194 (1996)), and DNA binding proteins (Beerli et al., Proc. Natl. Acad. Sci. USA 95:14628-14633 (1997); Liu et al., Proc. Natl. Acad. Sci. USA 94:5525-5530 (1998)).
Certain of these mechanisms are associated with nucleic acid homology at the DNA or RNA level (Matzke et al., Current Opinion in Genetics and Development 11:221-227 (2001)). In plants, double-stranded RNA molecules can induce sequence-specific silencing. This phenomenon is often referred to as double stranded RNA (“dsRNA”) in plants. This phenomenon has also been reported in Caenorhabditis elegans, where this gene-specific silencing is often referred to as RNA interference or RNAi (Fire et al., Nature 391:806-811 (1988). Others have reported this phenomenon in plants, fungi and animals (Sharp, Genes and Development 13:139-141 (1999); Matzke et al., Current Opinion in Genetics and Development 11:221-227 (2001); Cogoni and Macino, Current Opinion in Genetics and Development 10:638-643 (2000); Sharp, Genes and Development 15:485-490 (2001); Waterhouse et al., Proc. Natl. Acad. Sci. USA 95:13959-13964 (1988); Wesley et al., Plant J. 27:581-590 (2001); Grierson, WO 98/53083). Wesley et al. reported the design and use of two vectors, pHANNIBAL and pHELLSGATE, that can be used as gene silencing vectors (Wesley et al., supra). These vectors are reported to contain an intron sequence between the sense and antisense sequences where the sense and antisense sequences correspond to a target coding sequence, 5′UTR or 3′UTR. By utilizing a non-target intron between the target sense and antisense sequences, a higher proportion of silenced transformants were obtained (Wesley et al., supra). Another strategy of gene silencing with dsRNA involves a hairpin construct with an intron spacer (Smith et al., Nature 407:319-320 (2000)).
Other suppression strategies include, without limitation, antisense and sense suppression. See e.g. Fillatti in PCT WO 01/14538.
A desired plant phenotype may require the expression of one gene and the concurrent reduction of expression of another gene. Thus, there exists a need to simultaneously over-express a polypeptide and suppress, or down-regulate, the expression of a second polypeptide in plants using a single transgenic construct. Moreover, there exists a need to simultaneously suppress or down-regulate the expression of more than one polypeptide using a single construct.